The human papillomaviruses (HPVs) are causally linked to a number of human cancers. Over 120 different HPVs have been identified and a subset of them are associated with lesions that are at risk for progression to cancer. Virtually al cases of cervical cancer are attributable to infection by HPV. Of the many different HPV types, 14 are referred to as high risk because of their association with anogenital cancers. These high risk HPVs are associated with cervical cancer, other anogenital cancers and approximately 20% of head and neck cancers. The current VLP-based vaccines prevent infection by only 2 of the 14 high-risk HPV types and vaccination rates in young women vary greatly among countries; in the United States the rate of vaccination among young women is presently approximately only 30%. Despite the availability of VLP-based preventive vaccines against HPV16 and HPV18 (that account for roughly 70% of cervical cancers), there is a need for therapeutic options targeting HPV- associated cancers and the persistent infections that give rise to these cancers. With 500,000 new cases of cervical cancer diagnosed annually and current vaccines having no therapeutic benefit for the millions of already infected women, there is a need for the development of treatment options Two viral oncogenes, E6 and E7, have been implicated in driving HPV-associated carcinogenesis. The best characterized function of E6 is the targeting of p53 for degradation. E6 mediates p53 degradation by hijacking the cellular ubiquitin ligase E6-associated protein (E6AP) to form a complex that ubiquitylates p53. Inhibition of E6 or E6AP in HPV-positive cells leads to p53 stabilization and subsequent apoptosis due to E7-induced oncogenic stress, validating the inhibition of E6/E6AP activity as a potential therapeutic approach for treating HPV-associated cancers and persistent infections. The goal of this developmental application is to identify, via a high throughput screen, small molecules that stabilize p53 in HPV positive cervical cancer. We have engineered HPV16 and HPV18 cervical cancer cell lines to provide a GFP/DsRed fluorescent ratio indicator of p53 stability in living cells. The small molecule screen proposed will utilize the p53 reporter SiHa cells (an HPV16 positive cervical cancer cell line) and would employ a counter screen using a similarly engineered human osteosarcoma cell line (SJSA-1) in which p53 is regulated by Mdm2 rather than E6/E6AP to identify small molecules that would be specific for HPV-associated cancers and precancerous persistent infections. This developmental application brings together the laboratories of Peter Howley with expertise in HPV and Nathanael Gray with expertise in medicinal chemistry to address an important need in cancer treatment by identifying small molecules that could serve as tool compounds or even lead compounds for development of therapies to treat cervical cancer and HPV-positive preneoplastic lesions.